LKKTET and/or LKKTNT peptide compositions which are lyophilized or in a form capable of being lyophilized

ABSTRACT

A composition including a peptide agent including amino acid sequence LKKTET [SEQ ID NO: 1] or LKKTNT [SEQ ID NO: 2], a conservative variant thereof, or a stimulating agent that stimulates production of an LKKTET [SEQ ID NO: 1] or LKKTNT [SEQ ID NO: 2] peptide, or a conservative variant thereof, the composition including at least one amino acid stabilizing agent or lyophilization bulking agent, the composition being in lyophilized form, or in a form capable of being lyophilized.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a 35 U.S.C. §371 National Phase of InternationalApplication Serial No. PCT/US2006/023759, filed Jun. 19, 2006, andclaims benefit of U.S. Provisional Application No. 60/691,261, filedJun. 17, 2005 and U.S. Provisional Application No. 60/776,947, filedFeb. 28, 2006.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of LKKTET [SEQ ID NO:1]and/or LKKTNT [SEQ ID NO:2] compositions and methods.

2. Description of the Background Art

Thymosin beta 4 initially was identified as a protein that isup-regulated during endothelial cell migration and differentiation invitro. Thymosin beta 4 was originally isolated from the thymus and is a43 amino acid, 4.9 kDa ubiquitous polypeptide identified in a variety oftissues. Several roles have been ascribed to this protein including arole in a endothelial cell differentiation and migration, T celldifferentiation, actin sequestration, vascularization and wound healing.

Many Tβ4 isoforms have been identified and have about 70%, or about 75%,or about 80% or more homology to the known amino acid sequence of Tβ4.Such isoforms include, for example, Tβ4^(ala), Tβ9, Tβ10, Tβ11, Tβ12,Tβ13, Tβ14 and Tβ15. Similar to Tβ4, the Tβ10 and Tβ15 isoforms havebeen shown to sequester actin. Tβ4, Tβ10 and Tβ15, as well as theseother isoforms share an amino acid sequence, LKKTET [SEQ ID NO:1] orLKKTNT [SEQ ID NO:2], that appears to be involved in mediating actinsequestration or binding. Although not wishing to be bound by anyparticular theory, the activity of peptide agents as described hereinmay be due, at least in part, to the anti-inflammatory activity of suchagents. Tβ4 also can modulate actin polymerization (e.g. β-thymosinsappear to depolymerize F-actin by sequestering free G-actin). Tβ4'sability to modulate actin polymerization may be due to its ability tobind to or sequester actin via the LKKTET [SEQ ID NO:1] or LKKTNT [SEQID NO:2] sequence.

There remains a need in the art for LKKTET [SEQ ID NO:1] and/or LKKTNT[SEQ ID NO:2] compositions and methods.

SUMMARY OF THE INVENTION

According to one embodiment, a composition comprising a peptide agentcomprises amino acid sequence LKKTET [SEQ ID NO:1] or LKKTNT [SEQ IDNO:2], a conservative variant thereof, or a stimulating agent thatstimulates production of an LKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO:2]peptide, or a conservative variant thereof, the composition including atleast one amino acid stabilizing agent, the composition being inlyophilized form, or in a form capable of being lyophilized.

According to another embodiment, a composition comprising a peptideagent comprises amino acid sequence LKKTET [SEQ ID NO:1] or LKKTNT [SEQID NO:2], a conservative variant thereof, or a stimulating agent thatstimulates production of an LKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO:2]peptide, or a conservative variant thereof, and at least one of alyophilization bulking agent or an amino acid stabilizing agent, saidcomposition being in lyophilized form.

DETAILED DESCRIPTION OF THE INVENTION

Many compositions that are administered to subjects for various purposescontain one or more ingredients that may cause injury to the subject.

For example, preservatives such as benzalkonium chloride (BAK), whichare present in many ophthalmic and cosmetic products, may cause surfaceirritation and/or injury when the product is administered. Symptoms mayinclude redness and discomfort.

There is a significant and growing problem with ocular surfaceirritation and apoptosis with chronic use of eye drops containingquaternary ammonium salts such as BAK. Patients often develop symptomssuch as redness and general eye discomfort. A peptide agent inaccordance with the present invention, such as thymosin beta 4 (Tβ4),can be utilized to treat or prevent damage or injury to eye tissue whenadministered to a subject in combination with contacting the eye tissuewith a quaternary ammonium salt such as BAK.

Examples of quaternary ammonium salts to which the present invention maybe applicable include BAK, cetrimide, benzoxonium chloride, and thelike.

According to one embodiment, the peptide agent is administered togetherwith the quaternary ammonium salt such as BAK as part of the sameformulation, e.g., as an additive to a formulation containing BAK orother quaternary ammonium salt. Quaternary ammonium salts such as BAKare ingredients commonly used as preservatives in many ocular solutions,such as glaucoma eye drops.

In alternative embodiments, the peptide agent can be administered beforeadministration of the quaternary ammonium salt such as BAK, and/orduring administration of the quaternary ammonium salt such as BAK,and/or after administration of the quaternary ammonium salt such as BAK.

According to one embodiment the invention is particularly useful inpreventing damage to corneal epithelial eye tissue, including preventionof apoptosis of cells of such eye tissue. For example, the invention maybe utilized as an adjuvant for anti-glaucoma drops or other eye drops.

In accordance with one aspect, an ophthalmically acceptable compositioncomprises a peptide agent comprising amino acid sequence LKKTET [SEQ IDNO:1] or LKKTNT [SEQ ID NO:2], or a conservative variant thereof.

In accordance with another aspect, a method of treating eye tissuecomprises topically administering to said eye tissue an ophthalmicallyacceptable composition comprising a peptide agent comprising amino acidsequence LKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO:2], or a conservativevariant thereof.

In one embodiment, the invention provides a method of treatment bycontacting eye tissue with an effective amount of a composition whichcontains a peptide agent as described herein. Examples of directadministration include, for example, contacting the tissue, by directapplication with a solution, lotion, salve, gel, cream, paste, spray,suspension, dispersion, hydrogel, ointment, oil or foam comprising apeptide agent as described herein.

Without being found to any specific theory, actin-sequestering peptidessuch as thymosin beta 4 (Tβ4 or TB4) and other agents includingactin-sequestering peptides or peptide fragments containing amino acidsequence LKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO:2] or conservativevariants thereof, promote healthy eye tissue.

Thymosin beta 4 was initially identified as a protein that isup-regulated during endothelial cell migration and differentiation invitro. Thymosin beta 4 was originally isolated from the thymus and is a43 amino acid, 4.9 kDa ubiquitous polypeptide identified in a variety oftissues. Several roles have been ascribed to this protein including arole in a endothelial cell differentiation and migration, T celldifferentiation, actin sequestration, vascularization and wound healing.

According to one embodiment the invention is preferably applicable toThymosin β4, and/or Tβ34 isoforms, analogues or derivatives, includingKLKKTET [SEQ ID NO:3], LKKTETQ [SEQ ID NO:4], oxidized Tβ4, Tβ4sulfoxide, N-terminal variants of Tβ4 and C-terminal variants of Tβ4.

According to one embodiment, compositions which may be used inaccordance with the present invention include peptide agents such asThymosinβ4 (Tβ4), and/or Tβ4 isoforms, analogues or derivatives,including oxidized forms of Tβ4 including Tβ4 sulfoxide, N-terminalvariants of Tβ4, and C-terminal variants of Tβ4, and polypeptides orpeptide fragments comprising or consisting essentially of the amino acidsequence LKKTET [SEQ ID NO:1] and conservative variants thereof.International Application Serial No. PCT/US99/17282, incorporated hereinby reference, discloses isoforms of Tβ4 which may be useful inaccordance with the present invention as well as amino acid sequenceLKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO:2] and conservative variantsthereof, which may be utilized with the present invention. InternationalApplication Serial No. PCT/GB99/00833 (WO 99/49883), incorporated hereinby reference, discloses oxidized Thymosin β4 which may be utilized inaccordance with the present invention. Although the present invention isdescribed primarily hereinafter with respect to Tβ4 and Tβ4 isoforms, itis to be understood that the following description is intended to beequally applicable to amino acid sequence LKKTET [SEQ ID NO:1] or LKKTNT[SEQ ID NO:2], peptides and fragments comprising or consistingessentially of LKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO:2],conservative variants thereof and/or Tβ4 isoforms, analogues orderivatives, including oxidized Tβ4, N-terminal variants of Tβ4, andC-terminal variants of Tβ4.

According to one embodiment a composition in accordance with the presentinvention can be administered daily, every other day, every other week,every other month, etc., with a single application or multipleapplications per day of administration, such as applications 2, 3, 4 ormore times per day of administration.

Many Tβ4 isoforms have been identified and have about 70%, or about 75%,or about 80% or more homology to the known amino acid sequence of Tβ4.Such isoforms include, for example, Tβ4^(ala), Tβ9, Tβ10, Tβ11, T12,Trβ13, Tβ14 and Tβ15. Similar to Tβ4, the Tβ10 and Tβ15 isoforms havebeen shown to sequester actin. Tβ4, Tβ10 and Tβ15, as well as theseother isoforms share an amino acid sequence, LKKTET [SEQ ID NO:1] orLKKTNT [SEQ ID NO:2], that appears to be involved in mediating actinsequestration or binding. Tβ4 has anti-inflammatory activity, and alsocan modulate actin polymerization (e.g. β-thymosins appear todepolymerize F-actin by sequestering free G-actin). Tβ4's ability tomodulate actin polymerization may be due to its ability to bind to orsequester actin via the LKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO:2]sequence. Thus, as with Tβ4, other proteins which are anti-inflammatoryand/or bind or sequester actin, or modulate actin polymerization,including Tβ4 isoforms having the amino acid sequence LKKTET [SEQ IDNO:1], are likely to be effective, alone or in a combination with Tβ4,as set forth herein.

Thus, it is specifically contemplated that known Tβ4 isoforms, such asTβ4^(ala), Tβ9, Tβ10, Tβ11, Tβ12, Tβ13, Tβ14 and Tβ15, as well as Tβ4isoforms not yet identified, will be useful in the methods of theinvention. As such Tβ4 isoforms are useful in the methods of theinvention, including the methods practiced in a subject. The inventiontherefore further provides compositions comprising Tβ4, as well as Tβ4isoforms Tβ4^(ala), Tβ9, Tβ10, Tβ11, Tβ12, Tβ13, Tβ14 and Tβ15, and anophthalmically acceptable carrier.

In addition, other agents or proteins having anti inflammatory activityand/or actin sequestering or binding capability, or that can mobilizeactin or modulate actin polymerization, as demonstrated in anappropriate sequestering, binding, mobilization or polymerization assay,or identified by the presence of an amino acid sequence that mediatesactin binding, such as LKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO:2], forexample, can similarly be employed in the methods of the invention. Suchproteins may include gelsolin, vitamin D binding protein (DBP),profilin, cofilin, depactin, Dnasel, vilin, fragmin, severin, cappingprotein, β-actinin and acumentin, for example. As such methods includethose practiced in a subject, the invention further providespharmaceutical compositions comprising gelsolin, vitamin D bindingprotein (DBP), profilin, cofilin, depactin, Dnasel, vilin, fragmin,severin, capping protein, β-actinin and acumentin as set forth herein.Thus, the invention includes the use of an polypeptide comprising theamino acid sequence LKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO:2] andconservative variants thereof.

As used herein, the term “conservative variant” or grammaticalvariations thereof denotes the replacement of an amino acid residue byanother, biologically similar residue. Examples of conservativevariations include the replacement of a hydrophobic residue such asisoleucine, valine, leucine or methionine for another, the replacementof a polar residue for another, such as the substitution of arginine forlysine, glutamic for aspartic acids, or glutamine for asparagine, andthe like.

According to one embodiment, a composition in accordance with thepresent invention is an eye drop formulation.

According to one embodiment, a composition for use in the inventioncontains a peptide agent as described herein at a concentration within arange of from about 0.001-1000 micrograms per ml (mcg/ml), morepreferably about 0.1-100 mcg/ml, most preferably about 1-10 mcg/ml. Inparticularly preferred embodiments, the peptide agent is Tβ4.

According to one embodiment the invention is useful for treating orpreventing damage or injury to eye tissue resulting from contacting eyetissue with a quaternary ammonium salt such as BAK at concentrationswithin a range of about 0.0001-1% by weight, preferably within a rangeof about 0.001-0.1% by weight, and more preferably within a range ofabout 0.002-0.05% by weight. In one embodiment, the quaternary ammoniumsalt is at a concentration of about 0.005-0.02% by weight.

According to one embodiment the invention also includes a pharmaceuticalcomposition comprising a peptide agent as described herein in anophthalmically acceptable carrier. Such carriers include, e.g., thoselisted herein.

According to one embodiment the actual dosage or reagent, formulation orcomposition that provides treatment may depend on many factors,including the size and health of a subject. However, persons of ordinaryskill in the art can use teachings describing the methods and techniquesfor determining clinical dosages as disclosed in PCT/US99/17282, supra,and the references cited therein, to determine the appropriate dosage touse.

According to one embodiment the methods and compositions using orcontaining a peptide agent as described herein may be formulated intocompositions by admixture with ophthalmically acceptable non-toxicexcipients or carriers.

According to one embodiment the topical formulation containing theactive compound can also contain a physiologically compatible vehicle,as those skilled in the ophthalmic art can select using conventionalcriteria. The vehicles can be selected from the known ophthalmicvehicles which include, but are not limited to, saline solution, waterpolyethers such as polyethylene glycol, polyvinyls such as polyvinylalcohol and povidone, cellulose derivatives such as methylcellulose andhydroxypropyl methylcellulose, petroleum derivatives such as mineral oiland white petrolatum, animal fats such as lanolin, polymers of acrylicacid such as carboxypolymethylene gel, vegetable fats such as peanut oiland polysaccharides such as dextrans, and glycosaminoglycans such assodium hyaluronate and salts such as sodium chloride and potassiumchloride.

According to one embodiment an ophthalmic composition is advantageouslyapplied topically to the eye, especially in the form of a solution, asuspension, an ointment, gel or foam.

According to one embodiment there are used for a correspondingophthalmic composition customary pharmaceutically acceptable excipientsand additives known to the person skilled in the art, for example thoseof the type mentioned below, especially carriers, stabilizers,solubilizers, tonicity enhancing agents, buffer substances,preservatives, thickeners, complexing agents and other excipients.Examples of such additives and excipients can be found in U.S. Pat. Nos.5,134,124 and 4,906,613. Such compositions are prepared in a mannerknown per se, for example by mixing the active ingredient with thecorresponding excipients and/or additives to form correspondingophthalmic compositions. The active ingredient is preferablyadministered in the form of eye drops, the active ingredient beingconventionally dissolved, for example, in a carrier. The solution is,where appropriate, adjusted and/or buffered to the desired pH and, whereappropriate, a stabilizer, a solubilizer or a tonicity enhancing agentis added. Where appropriate, preservatives and/or other excipients areadded to an ophthalmic composition.

Carriers used in accordance with the present invention are typicallysuitable for topical or general administration, and are for examplewater, mixtures of water and water-miscible solvents, such asC₁-C₇-alkanols, vegetable oils or mineral oils comprising from 0.5 to 5%by weight hydroxyethylcellulose, ethyl oleate, carboxymethylcellulose,polyvinyl-pyrrolidone and other non-toxic water-soluble polymers forophthalmic uses, such as, for example, cellulose derivatives, such asmethylcellulose, alkali metal salts of carboxymethylcellulose,hydroxymethylcellulose, hydroxyethylcellulose,methylhydroxypropylcellulose and hydroxypropylcellulose, acrylates ormethacrylates, such as salts of polyacrylic acid or ethyl acrylate,polyacrylamides, natural products, such as gelatin, alginates, pectins,tragacanth, karaya gum, xanthan gum, carrageenin, agar and acacia,starch derivatives, such as starch acetate and hydroxypropyl starch, andalso other synthetic products, such as polyvinyl alcohol,polyvinylpyrrolidone, polyvinyl methyl ether, polyethylene oxide,preferably cross-linked polyacrylic acid, such as neutral Carbopol, ormixtures of those polymers. Preferred carriers are water, cellulosederivatives, such as methylcellulose, alkali metal salts ofcarboxymethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose,methylhydroxypropylcellulose and hydroxypropylcellulose, neutralCarbopol, or mixtures thereof.

According to one embodiment the solubilizers used for an ophthalmiccomposition of the present invention are, for example, tyloxapol, fattyacid glycerol poly-lower alkylene glycol esters, fatty acid poly-loweralkylene glycol esters, polyethylene glycols, glycerol ethers ormixtures of those compounds. The amount added is typically sufficient tosolubilize the active ingredient. For example, the concentration of thesolubilizer is from 0.1 to 5000 times the concentration of the activeingredient. Lower alkylene means linear or branched alkylene with up toand including 7 C-atoms. Examples are methylene, ethylene,1,3-propylene, 1,2-propylene, 1,5-pentylene, 2,5-hexylene or1,7-heptylene. Lower alkylene is preferably linear or branched alkylenewith up to and including 4 C-atoms.

Examples of buffer substances are acetate, ascorbate, borate, hydrogencarbonate/carbonate, citrate, gluconate, lactate, phosphate, propionateand TRIS (tromethamine) buffers. Tromethamine and borate buffer arepreferred buffers. The amount of buffer substance added is, for example,that necessary to ensure and maintain a physiologically tolerable pHrange. The pH range is typically in the range of from 5 to 9, preferablyfrom 6 to 8.2 and more preferably from 6.8 to 8.1.

Tonicity enhancing agents are, for example, ionic compounds, such asalkali metal or alkaline earth metal halides, such as, for example,CaCl₂, KBr, KCl, LiCl, NaBr, NaCl, or boric acid. Non-ionic tonicityenhancing agents are, for example, urea, glycerol, sorbitol, mannitol,propylene glycol, or dextrose. For example, sufficient tonicityenhancing agent is added to impart to the ready-for-use ophthalmiccomposition an osmolality of approximately from 50 to 1000 mOsmol,preferred from 100 to 400 mOsmol, more preferred from 200 to 400 mOsmoland even more preferred from 280 to 350 mOsmol.

Examples of preservatives are quaternary ammonium salts, such ascetrimide, benzalkonium chloride or benzoxonium chloride, alkyl-mercurysalts of thiosalicylic acid, such as, for example, thimerosal,phenylmercuric nitrate, phenylmercuric acetate or phenylmercuric borate,parabens, such as, for example, methylparaben or propylparaben,alcohols, such as, for example, chlorobutanol, benzyl alcohol or phenylethanol, guanidine derivatives, such as, for example, chlorohexidine orpolyhexamethylene biguanide, or sorbic acid. Preferred preservatives arecetrimide, benzalkonium chloride, benzoxonium chloride and parabens.Where appropriate, a sufficient amount of preservative is added to theophthalmic composition to ensure protection against secondarycontaminations during use caused by bacteria and fungi.

According to one embodiment the ophthalmic compositions may comprisefurther non-toxic excipients, such as, for example, emulsifiers, wettingagents or fillers, such as, for example, the polyethylene glycolsdesignated 200, 300, 400 and 600, or Carbowax designated 1000, 1500,4000, 6000 and 10 000. Other excipients that may be used if desired arelisted below but they are not intended to limit in any way the scope ofthe possible excipients. They are especially complexing agents, such asdisodium-EDTA or EDTA, antioxidants, such as ascorbic acid,acetylcysteine, cysteine, sodium hydrogen sulfite, butyl-hydroxyanisole,butyl-hydroxy-toluene or α-tocopherol acetate; stabilizers, such as acyclodextrin, thiourea, thiosorbitol, sodium dioctyl sulfosuccinate ormonothioglycerol; or other excipients, such as, for example, lauric acidsorbitol ester, triethanol amine oleate or palmitic acid ester.Preferred excipients are complexing agents, such as disodium-EDTA andstabilizers, such as a cyclodextrin. The amount and type of excipientadded is in accordance with the particular requirements and is generallyin the range of from approximately 0.0001 to approximately 90% byweight. A cyclodextrin is composed of several glucose units which havethree free hydroxy groups per glucose. The amount of a cyclodextrin usedin accordance with one embodiment may preferably range from 0.01-20% byweight, more preferably from 0.1-15% by weight and even more preferablyfrom 1-10% by weight.

According to one embodiment the present invention relates also to anophthalmic composition, which comprises a therapeutically effectiveamount of a peptide agent as described herein a carrier, a solubilizerand another therapeutically effective pharmaceutical agent which may be,for example, an antibiotic, an antiallergic, an anesthetic, anotherantiphlogistic, a corticosteroide, an agent suitable for loweringintra-ocular pressure, or another drug.

pH (Hydrogen Ion Concentration)

According to one embodiment, the pH of the inventive formulations shouldbe as close to that of the tear film as possible. The physiologic pH oftears is approximately 7.4±0.2. Thus, from a comfort, tolerability andsafety perspective, this would be the optimal pH of ophthalmicpreparations.

Stimulation of tear secretion and eye blinking causes the pH to decreasein value. When the eyelid remains open for extended periods of time thetear-film is alkalized by equilibrium with the partial pressure of CO₂in the surrounding air and a pH value of greater than 9 is attained.Both decreases and increases in pH occur without adverse consequences.Thus, there is some latitude in the pH range when formulating inventiveformulations around pH of approximately 7.4.

Also, when a formulation is administered to the eye, it stimulates theflow of tears. Tear fluid is capable of quickly diluting and bufferingsmall volumes of added substances, suggesting the eye can tolerate afairly wide pH range offered by certain formulations.

Consequently, ophthalmic formulations may be within a range of fromabout pH 3.5 to 11.5. However, ophthalmic formulations may display pHranges somewhat more narrowly from 3.5 to 9, preferably from 4.5 to 8and most preferably from pH 5.5 to 7.8. The most preferred pH range isadvantageous from the perspective solubility, chemical stability andtherapeutic activity of the inventive compositions and a useful andrelatively narrow range to prevent corneal damage.

Buffer Systems

According to one embodiment buffer systems are composed of a weak acidor base and its conjugate salt. The buffering capacity of the componentsin the system acts in such a way that, despite the addition of an acidor base and exposure to external influences of temperature, pressure,volume, redox potential, body fluids and tears, the pH will remainessentially constant. Although buffer capacity should be large enough toresist changes in the product pH (i.e., pH drift) for a reasonableshelf-life (i.e., under storage conditions), the buffer capacity ofinventive ophthalmic formulations should be low enough to allow rapidreadjustment of the product to physiologic pH upon administration to theeye. According to one embodiment buffer capacities for ophthalmicproducts should be within the range of 0.05 to 1.0. Preferred and mostpreferred buffer capacities range from 0.02 to 0.2 and 0.01 to 0.1,respectively for certain inventive compositions. Buffer capacity isdetermined by the following formula:*β=ΔB/ΔpHwhere β is buffer capacity, ΔB is the gram equivalent of strongacid/base to change pH of 1 liter of buffer solution and ΔpH is thechange caused by the addition of strong acid/base.

According to one embodiment appropriate buffer systems may be sodiumsalts of the following acids: acetic; ascorbic; boric; carbonic;phosphoric; citric; gluconic; lactic; and propionic. Calcium salts ofcarbonic or propionic acid form appropriate buffer systems as dopotassium salts of phosphoric acid. Tris buffer (tromethamine) is usedintravenously as an alkalizer for the correction of metabolic acidosisand is one of the preferred buffers for use in this invention. Otherpreferred buffers are acetate, phosphate, citrate and borate. In certaininstances a buffer system involving proton donor and proton acceptorgroups of the amino acid residues of proteins may be preferable to theacid-base or an amine-base buffer.

The specific amount of a buffer substance used will vary and dependsupon the amount that is deemed necessary to maintain a pH-environmentsuitable for the stability of inventive composition and to ensure andmaintain a physiologically tolerable pH range.

Tonicity Agents

Of those listed herein the objective is to adjust the tonicity of theinventive ophthalmic compositions to that of natural tears toapproximate physiological tonicity (e.g., 0.9% saline). For example,sodium chloride, potassium chlorides, calcium chloride, dextrose and/ormannitol may be added to the inventive peptide agent formulation. Theamount of tonicity agent will vary, depending on the particular agent tobe added. In general, certain inventive compositions will have atonicity agent in an amount sufficient to cause the final composition tohave an ophthalmically acceptable osmolality preferably 150-450 mOsm andmost preferably 250-350 mOsm.

The preferred tonicity agents are sodium salts and potassium salts,particularly sodium and potassium chloride. The most preferred tonicityagent is sodium chloride.

Lubricants/Demulcents/Viscosity Enhancers

According to one embodiment compounds may be included which sooth theeye, reduce surface tension and improve wettability (contact) of anotherwise hydrophobic epithelial corneal surface, approximate theconsistency of tears. Such compounds may also enhance the viscosity ofthe inventive compositions, allowing an inventive formulation to remainin the eye longer thus giving the peptide agent more time to exert itstherapeutic activity or undergo absorption to reach the desired target.

Suitable viscosity enhancers in ophthalmic formulations and theirconcentration ranges used in certain inventive compositions include butare not limited to: (a) Monomeric polyols, such as tyloxapol (0.1-1%),glycerol (0.2-1%), propylene glycol (0.2 to 1%), ethylene glycol(0.2-1%); (b) Polymeric polyols, such as polyethylene glycol (e.g., PEG300, PEG 400) (0.2-1%); (c) Cellulose derivatives (polymers of thecellulose family), such as hydroxyethylcellulose (0.2-2.5%),hypromellose (0.2 to 2.5%), hydroxypropylmethyl cellulose (0.2-2.5%),methylcellulose (0.2-2.5%), carboxymethylcellulose sodium (0.2 to 2.5%),hydroxylpropylcellulose (0.2-2.5%); (d) Dextrans, such as dextran 70(0.1% when used with another polymeric demulcent agent); (e)Water-soluble proteins such as gelatin (0.01%); (f) Vinyl polymers suchas polyvinyl alcohol (0.1-4%), polyvinyl pyrollidine (0.1-4%); (g) Otherpolyols, such as polysorbate 80 (0.2-1%), povidone (0.1-2%); (h)Carbomers, such as carbomer 934P, carbomer 941, carbomer 940, andcarbomer 974P, and (i) Polysaccharides/Glycosaminoglycans, such ashyaluronan (hyaluronic acid/hyaluronate) (0.1-3%), chondroitin sulfate(0.1-3%).

More than one viscosity enhancer may be added to an inventivecomposition to increase the viscosity of the carrier (vehicle). Apreferred enhancer in the carrier of an inventive peptide agentformulation is carboxymethylcellulose.

Viscosity

Viscosity describes a material's internal resistance to flow or changein form, when a stress is applied. The viscosity of a material(solution, semi-viscous gel, suspension, oleaginous ointments andointment gels (viscous gels) is given in poise units. The unit,centipoise (“cp” or the plural “cps”) is equal to 0.01 poise and is mostoften used in pharmaceutical applications. Compounds used to enhanceviscosity are available in various grades such as 15 cps, 100 cps, etc.,etc. The grade number refers to the viscosity which results when a fixedpercentage aqueous solution of the enhancer is made. Generally,solutions are 1% or 2%; however, they can be as high as 4% with certainenhancers. Viscosity is measured at 200 or 25° C.

A suitable viscosity in an ophthalmic solution is between 25 and 50centipoises (cps). The actual concentration of an enhancer required toproduce that desired viscosity will depend on the grade of the enhancer.For example, if methycellulose 25 cps is used, a 1% solution will createa viscosity of 25 cps. If methycellulose 4000 cps is used, an 0.25%solution provides the desired viscosity. Standard references give tablesof viscosities produced by percentage solutions and grades ofingredients.

According to one embodiment inventive formulations will exhibit aviscosity of >1 to 100,000 centipoises (cps) or greater. Inventiveointment compositions (oleaginous or viscous gels) may have viscositygrades that are greater than 100,000 cps. This is because ophthalmicointments are intended to be thick when standing to prevent them fromflowing away from the intended area of use. Following application andover time, temperatures within the conjunctival sac, or on the surfaceof the eye, where these ointments are deposited, will cause theseointments to “melt” and begin to flow.

The preferred viscosity ranges of various inventive formulation typesare found in the table below:

Formulation Type Viscosity Range (cps) Aqueous Solution   >1 to <1000Suspension   >1 to <1000 Gel    >3 to 40,000 Ointment, gel >20,000 to100,000 Ointment, oleaginous >20,000 to 100,000

Reducing Agents/Antioxidants/Oxygen-Sequestering Agents

Certain inventive compositions have the potential to be degraded byoxidation. Consequently, steps during the manufacture, control andpackaging of an inventive composition may include protecting inventivecompositions, susceptible to oxidation, by (1) displacing oxygen withnitrogen or a dense inert gas such as argon, (2) adding a reducing agentto minimize oxidative effects, (3) the introduction of a decoy molecule.

Common antioxidant (reducing) agents which may be used in ophthalmicformulations up to a concentration of 0.1% or more are sodium sulfite,sodium thiosulfite, sodium bisulfite, sodium metabisulfite, andthiourea. Sulfites can cause allergic-type reactions in certain people;consequently, patients receiving this type of antioxidant should bequestioned about this potential reaction before being treated with aninventive composition containing the antioxidant. Other usefulantioxidants compatible with the inventive compositions are ascorbicacid, EDTA/disodium edetate, acetic acid, citric acid, glutathione andacetylcysteine. These agents may also be regarded as stabilizers.

A decoy molecule or an oxygen sequestering protective agent may be addedas stabilizers to an inventive formulation to minimize oxidative effectson the inventive formulation. The molecular decoy must have at least thesame capability of being oxidized as the inventive formulation. One suchdecoy, for an inventive composition containing methionine is the aminoacid, methionine, itself. Free methionine added to an inventivecomposition containing the amino acid methionine would compete foroxygen in the process of being oxidized to methionyl sulfoxide. A freeoxygen-consuming agent is one that prevents other oxygen-reactive aminoacids in the inventive composition/peptide from being oxidized. For thepurposes of certain inventive compositions but not limited to such, afree oxygen-consuming agent is methionine.

Ophthalmic Ointments/Oleaginous Emollient Bases

Ophthalmic ointments tend to keep an active agent in contact with theeye longer than suspensions and certainly solutions. Most ointments,tend to blur vision, as they are not removed easily by the tear fluid.Thus, ointments are generally used at night as adjunctive therapy to eyedrops used during the day.

Oleaginous ointment bases of inventive compositions are mixtures ofmineral oil, petrolatum and lanolin all have a melting point close tobody temperature. In the case of the inventive compounds, thecompositions may include mineral oil, petrolatum or lanolin. Accordingto one embodiment preferred compositions would include a combination ofpetrolatum, mineral oil and lanolin. The most preferred composition isan ointment combination containing white petrolatum, mineral oil andlanolin (anhydrous).

The peptide agent comprising amino acid sequence LKKTET [SEQ ID NO:1] orLKKTNT [SEQ ID NO:2] or a conservative variant thereof is dissolved in asmall amount of purified water or 0.9% saline to affect dissolution.This aqueous solution is incorporated into anhydrous lanolin and thethen “liquid” lanolin (up to 10%) is mixed with the remainingointment/oleaginous emollient base ingredients, mineral oil (up to 30%)and white petrolatum (up to 60%).

Ophthalmic ointment tubes are typically small holding approximately 1-5grams of ointment, preferably 3.5 grams, and fitted with narrow gaugetips which permit the extrusion of narrow bands of ointment measured ininches or fractions thereof for dosing purposes.

Preservatives

Sterility is an absolute requirement of all ophthalmic formulations.Contaminated formulations may result in eye infections that couldultimately cause blindness, especially if the P. aeruginosa microbe isinvolved. Therefore, ophthalmic formulations as described herein must beprepared using techniques, unique for solutions, gels, suspensions andointments of the inventive compositions that assure sterility. Sterileformulations must be packaged in sterile containers. Most topicalophthalmic products are typically packaged in multidose form. As suchpreservatives are required to prevent microbial contamination of anotherwise sterile product during use. Suitable preservatives include:Quaternary ammonium compounds (salts), such as benzalkonium chloride(0.001 to 0.02%), benzethonium chloride, cetalkonium chloride,cetrimide, benzododecinium bromide and benzoxonium chloride;Alkyl-mercury salts of thiosalicylic acid, such as thimerosal (0.001 to0.005%); Parabens, such as methylparaben and propylparaben; Chelatingagents, such as disodium edetate, sodium gluconate, sodium propionate;Other agents, such as chlorobutanol, boric acid, sorbic acid,phenylethanol (0.25%); Purite® chlorine dioxide; Polyquad®polyquatemium-1 (0.001%); and Aldox® myristamidopropyl diethylamine(0.005%); or other agents known to those skilled in the art.

Such preservatives are typically employed at a level of from 0.001% to1.0% (w/v) to ensure protection against secondary microbialcontaminations during use caused by bacteria, mold, and fungi.

Maximum concentrations of the following selected preservatives,presently approved for use in ophthalmic formulations, are shown in thetable below:

Maximum Concentration Agent % Benzalkonium chloride (BAK) 0.01Benzethonium chloride 0.01 Clorobutanol 0.5 Phenylmercuric acetate 0.004Phenylmercuric nitrate 0.004 Thimerosal (thiomersal) 0.01 Methyparaben0.2 Propylparaben 0.04 Source: FDA Advisory Panel on OTC Opthalmic DrugProducts, Final report. Dec. 1979Source: FDA Advisory Panel on OTC Opthalmic Drug Products, Final report.December 1979

Selection of the appropriate preservative is based upon itsanti-microbial effectiveness with the chosen inventive composition.Preferred preservatives for use in the inventive formulations are thecombination of methylparaben (0.080%-0.1%) and propylparaben(0.016%-0.024%), benzalkonium chloride (BAK) (0.005%-0.02%, where 0.01%w/v is most preferred), a combination of BAK and EDTA (0.01-0.5%), whichwhen used together have synergistic effects.

Unit dose compositions of the present invention will be sterile butunpreserved. Such compositions for the most part will not containpreservatives. Consequently, these compositions cannot be re-used andonce-opened they must be discarded.

Bulking/Stabilizing Agents

Bulking/stabilizing agent(s) may be advantageous to maintain thehydration state of a lubricant, emollient or vehicle enhancer comprisingan inventive composition during long-term storage. Associations appearto occur within or among polymer chains of these substances which aftertime favor the reduction of hydration state of these chains. Theseassociations may be in the form of hydrogen bonds within and among thepolymer chains which can manifest as a change in viscosity and textureof an ophthalmic formulation/composition in the present invention.Lyophilization bulking agents, which are principally sugars, may also beconsidered stress protectants, protecting compounds during thelyophilization cycle. Agents which greatly decelerate or eliminate thisreduced state of hydration are a class of stabilizing orhydration-enhancing agents, the polyols at concentrations of 0.2 to 5%by weight. Representatives of such polyols are mannitol, sorbitol,glycerol, sucrose, related sugars, and the like. A most preferredstabilizing agent is the hydroscopic mannitol at concentrations rangingfrom 0.2% to 5% by weight.

Additionally, 50 mM amino acid stabilizers such as alanine (Ala), lysine(Lys), glycine (Gly) and glutamic acid (Glu) have been incorporated intothe formulated peptide agent containing sequence LKKTET [SEQ ID NO:1] orLKKTNT [SEQ ID NO:2] or a conservative variant thereof to improverecovery from reconstituted aqueous solutions following lyophilization.The preferred amino acid stabilizers are arginine and glycine, while themost preferred 50 mM amino acid is glycine.

Inventive Peptide Administration

Exemplary Topical Delivery (for Surface-Acting Effects)

Peptide agents comprising amino acid sequences LKKTET [SEQ ID NO:1] orLKKTNT [SEQ ID NO:2] or a conservative variant thereof are administeredto the surface of the eye for local effects to treat, for example:

-   -   1. Corneal epithelial wounds caused by but not limited to        chemical burns, recurrent corneal erosions, epithelial        debridement during surgery, corneal resurfacing procedures,        Laser-assisted In Situ Keratomileusis (LASIK);    -   2. Corneal epithelial thinning caused by quaternary ammonium        salts, such as BAK and the like;    -   3. Ocular inflammation (alone or in combination with        corticosteroids) to treat, for example, conjunctivitis,        blepharitis, keratitis, uveitis, scleritis, retinitis, optic        neuritis, and temporal arteritis;    -   4. Microbial infection (alone or in combination with        antibacterial, antifungal, or antiviral agents or in combination        with both antimicrobials and anti-inflammatory agents);    -   5. Dry eye syndrome (xerophthalmia);    -   6. Red eye [alone or in combination with ocular decongestants        (adrenergic vasoconstrictors of the conjunctiva), such as        ephedrine, naphazoline, phenylephirine, tetrahydrozoline and        antihistamines, such as pheniramine maleate) to whiten the eye];    -   7. Elevated intraocular pressure (IOP) and Glaucoma; and    -   8. Inflammatory or irritative conditions after traumatic injury        or surgery or in various eye irritation disorders

Topical inventive peptide agents are formulated as solutions,suspensions, gels and ointments. Inventive peptide agent formulationsmay be administered directly or indirectly by collagen sponges, insertsor the like. Every ophthalmic product, including topical ophthalmics,should be sterile in its final container to prevent microbialcontamination of the eye. Preservatives are added to the formulationwhen packaged in a multidose container for more than one use to maintainsterility once the container has been opened. Ophthalmic formulationsrequire that the pH, buffer capacity, viscosity and tonicity of theformulation are carefully controlled. Preferred pH ranges, buffers,viscosities and tonicities have been described herein.

Exemplary Formulation Topical Solution for Eye Drops

Each milliliter of a topically-applied inventive peptide formulationcontains the following peptide agent comprising amino acid sequenceLKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO:2] or a conservative variantas shown below:

Peptide at a concentration with a range of from about 0.001 to 1,000mg/ml, preferably from about 0.01 mg/ml to 600 mg/ml, more preferablypeptide at a concentration of about 0.1 mg/ml to 60 mg/ml, mostpreferably peptide at a concentration of about 1 mg/ml to 6 mg/ml.

Preferred Carrier/Vehicle: 20 mM Sodium citrate; 50 mM glycine; 3%sucrose; NaOH or HCl to adjust pH; Purified water, USP.

Exemplary Formulation Topical Suspension for Eye Drops

With regard to ophthalmic suspensions containing a peptide agentcomprising amino acid sequence LKKTET [SEQ ID NO:1] or LKKTNT [SEQ IDNO:2], particles must be less than 10 microns in size to minimizeirritation to the eye. There may be a tendency of the solid undissolvedparticles to adhere to the conjunctiva. As drug is absorbed, theseparticles will dissolve to replenish the absorbed drug. This reservoiror depot effect increases the contact time and duration of action of asuspension compared to a solution.

Each milliliter of a topically-applied inventive peptide formulationcontains the following peptide agent comprising amino acid sequenceLKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO:2] or a conservative variantas shown below:

Peptide at a concentration with a range of from about 0.001 to 1,000mg/ml, preferably from about 0.01 mg/ml to 600 mg/ml, more preferablypeptide at a concentration of about 0.1 mg/ml to 60 mg/ml, mostpreferably peptide at a concentration of about 1 mg/ml to 6 mg/ml.

Preferred Carrier/Vehicle: Peptide-encapsulation inpoly(lactide-co-glycolide) PLGA microspheres; 20 mM Sodium citrate; 50mM glycine; 3% sucrose; NaOH or HCl to adjust pH; Purified water, USP.

Exemplary Formulation Topical Gel for Eye Drops

Each milliliter of a topically-applied inventive peptide formulationcontains the following peptide agent comprising amino acid sequenceLKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO:2] or a conservative variantas shown below:

Peptide at a concentration of from about 0.001 to 1,000 mg/ml,preferably from about 0.01 mg/ml to 600 mg/ml, more preferably peptideat a concentration of about 0.1 mg/ml to 60 mg/ml, most preferablypeptide at concentration of about 1 mg/ml to 6 mg/ml.

Preferred Carrier/Vehicle: carboxymethylcellulose sodium (0.5 to 1%);dibasic sodium phosphate; sodium chloride; propylene glycol;methylparaben; propylparaben; NaOH/HCl to adjust pH; Purified water,USP.

Exemplary Formulation Topical Ointments

Each milliliter of a topically-applied inventive peptide formulationcontains the following peptide agent comprising amino acid sequenceLKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO:2] or a conservative variantas shown below:

Peptide at a concentration of from about 0.001 to 1,000 mg/ml,preferably from about 0.01 mg/ml to 600 mg/ml, more preferably peptideat concentration of about 0.1 mg/ml to 60 mg/ml, most preferably peptideat concentration of about 1 mg/ml to 6 mg/ml.

Preferred Carrier/Vehicle (1): carboxymethycellulose sodium (2.5%);dibasic sodium phosphate; propylene glycol; methylparaben;propylparaben; sodium chloride; NaOH/HCl to adjust pH; purified water.

Preferred Carrier/Vehicle (2): “liquid” lanolin (10%); mineral oil(30%), and white petrolatum (60%).

Exemplary Topical Delivery for Steroid-Sparing Effects

Corticosteroids inhibit the inflammatory response to a variety ofinciting agents.

-   -   Dexamethasone ophthalmic suspension (0.1%); Dexamethasone        ophthalmic ointment (0.05%); and Dexamethasone Sodium Phosphate        Ophthalmic Solution (0.1%)    -   Fluorometholone Ophthalmic Ointment (0.1%); Fluorometholone        Ophthalmic Suspensions (0.25-1%); and Fluorometholone Acetate        Ophthalmic Suspension (0.1%)    -   Lotoprednol etabonate (0.5%)    -   Medrysone Ophthalmic Suspension (1%)    -   Prednisolone Acetate Ophthalmic Suspensions (0.12-1%) and        Prednisolone Sodium Phosphate Ophthalmic Solutions (0.125-1%)    -   Rimexolone Ophthalmic Suspension (1%)

These agents, however, can elevate intraocular pressure (IOP) and, insusceptible individuals, can induce glaucoma with damage to the opticnerve, defects in visual acuity and fields of vision, and posteriorsubcapsular cataract formation. Cataract formation is a complicationmore likely to occur with high-dose, longterm use. Somecorticosterioids, such as fluorometholone acetate, medrysone, andloteprednol cause less elevation of IOP than others. Prolonged use mayalso suppress the host immune response and thus aid in the establishmentof secondary ocular infections from fungi and viruses liberated fromocular tissue. Topical corticosteroids are known to delay or slow woundhealing.

Administration of topically-applied eye drops or ointments containing aninventive peptide agent comprising amino acid sequence LKKTET [SEQ IDNO:1] or LKKTNT [SEQ ID NO:2] or a conservative variant to inhibit aninflammatory response to an inciting agent has the potential to besteroid-sparing.

Exemplary Intraocular Drug Delivery—Conjunctival/Sclera Instillation

The topical conjunctival route of entry plays an important role in thepenetration of drugs into the anterior segment. Furthermore, topicallyapplied drugs have been shown to have access to the sclera from theconjuctiva. The potential for transport or diffusion through the scleralies in the large and accessible surface area of this tissue, with itshigh degree of hydration, hypocellularity, and permeability that do notdecline significantly with age. As such, it is conceivable thatinventive compositions could find their way to the posterior segment bythis noninvasive route of administration. Data suggest that the sclerais readily permeable to even large molecular weight compounds (˜150 kD),much larger than a peptide agent comprising amino acid sequence LKKTET[SEQ ID NO:1] or LKKTNT [SEQ ID NO:2] or a conservative variant thereof.The recent finding that topically-applied nepafenac inhibited choroidaland retinal neovascularization by decreasing the production of VEGF, anda large molecular weight peptide like insulin (5.8 kD) can accumulate inthe retina and optic nerve after topical administration indicates thattopically applied inventive compositions, all of which having molecularweights of <150 kD, could not only reach the posterior segment throughconjunctival penetration, but that they can also be therapeutic. Topicalsolutions, suspensions, gels or ointments of peptide agents comprisingamino acid sequences LKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO:2] orconservative variants described above are suitable formulations fortopical conjuctival and scleral application.

Additionally, subconjuctival administration by injection of inventivecompositions of the peptide agent comprising amino acid sequence LKKTET[SEQ ID NO:1] or LKKTNT [SEQ ID NO:2] or a conservative variant thereofis useful in delivering anti-inflammatory and anti-microbial regimens,sensitive to the inventive composition, to treat serious ocularinflammation and ocular infections, such as uveitis and endophthalmitis,and glaucoma.

Preferred Injectable Formulation: Each ml of a peptide agent comprisingamino acid sequence LKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO:2] or aconservative variant contains:

Peptide at a concentration of from about 0.001 to 1,000 mg/ml,preferably from about 0.01 mg/ml to 600 mg/ml, more preferably peptideat a concentration of about 0.1 mg/ml to 60 mg/ml, most preferablypeptide at a concentration of about 1 mg/ml to 6 mg/ml.

Preferred Carrier/Vehicle: 20 mM Sodium citrate; 50 mM glycine; 3%sucrose; NaOH or HCl to adjust pH; Water for Injection, USP.

Exemplary Intraocular Drug Delivery—Transcorneal Instillation

Topically applied drugs do penetrate into the intraocular environmentthrough the hydrophobic cornea; however, transcorneal transport is notthe most effective process as it is estimated that only one- tothree-tenths of a dose penetrates into the eye while most of the drugremains confined to the superficial epithelium layer. Passive diffusionof inventive peptide compositions across the cornea is largelyinfluenced by their solubility, molecular weight and degree ofionization. Having a net negative charge and a relatively high molecularweight, an inventive peptide agent, formulated as a topically-applieddrug will find it difficult to penetrate the intact cornea. This issupported by the fact that pores, localized between epithelial cells inthe cornea allow paracellular permeation of only small molecules ofabout 500-molecular weight or less. However, in cases where the intactcorneal epithelium has been disrupted by erosion, for example, orexposed to a substance or penetrating agent that opens tight junctionsbetween epithelial cells, inventive compositions will pass through thecornea into the intraocular space more efficiently.

Topical solutions, gels or ointments of peptide agents comprising aminoacid sequences LKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO:2] orconservative variants described above are suitable formulations fortranscorneal instillation.

Exemplary Intraocular Drug Delivery—Periocular Injection

Periocular injection formulations of inventive peptide agents are usedin cases where ocular inflammation is not responding to a topical eyedrops alone and in inflammatory conditions such as anterior uveitis,posterior uveitis, endophthalmitis, and optic neuritis. The peptideagent is injected just below the conjunctiva or in the space belowTenon's capsule. Here, more absorption will occur and consequently, moredrug will be available to the desired site. Periocular injection isadditive to topical therapy but lacks the convenience and is notwell-tolerated to be considered a first line treatment.

Preferred Injectable Formulation: Each ml of the peptide agentcomprising amino acid sequence LKKTET [SEQ ID NO:1] or LKKTNT [SEQ IDNO:2] or a conservative variant contains:

Peptide at a concentration of from about 0.001 to 1,000 mg/ml,preferably from about 0.01 mg/ml to 600 mg/ml, more preferably peptideat a concentration of about 0.1 mg/ml to 60 mg/ml, most preferablypeptide at a concentration of about 1 mg/ml to 6 mg/ml.

Preferred Carrier/Vehicle: 20 mM Sodium citrate; 50 mM glycine; 3%sucrose; NaOH or HCl to adjust pH; Water for Injection, USP.

Exemplary Intraocular Drug Delivery—IntraVitreal/IntraAqueousAdministration

As an alternative to transcorneal, transconjuctival, and transscleraltransport, delivery of inventive peptide agents to the intraoculartissues can be achieved by injection into the vitreous or aqueouscavitiy. The vitreous is made of a hydrogel (water, hyaluronic acid andcollagen), which fills the cavity between the retina and the lens, whilethe aqueous is a watery fluid which fills the cavity between the lensand the iris. Intravitreal or intra-aqueous injections of an inventivepeptide agent, formulated as a solution allows for immediate exposure ofintraocular tissues to the peptide agent. To achieve a continuousintraocular presence of an inventive agent, which may be quicklyeliminated from the vitreous, would require repeated injections thatincrease the risk of endophthalmitis, damage to lens, retinal detachmentand may be poorly tolerated. To obviate this obstacle, the inventivepeptide agent would be encapsulated within phospholipid membranes, i.e.,liposomes, biodegradable microspheres, nanoparticles, or biodegradablelactone based polymers that includes polyesters made by polycondensationof L-lactide, glycolide, caprolactone, dioxanone, cyclic carbonates andtheir derivatives. Polylactide and polyglycolide, known also aspoly(lactic-acid) PLA and poly(glycolic-acid) PGA, respectively, andparticularly their co-polymers poly(lactide-co-glycolide) PLGA are themost investigated biodegradable polymers, which also can also be appliedas carriers for the inventive peptide agents. Additionally,peptide-polymer conjugates such as the covalent linkage of the peptideagent comprising LKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO:2] or aconservative variant thereof with synthetic and natural polymers such aspolyethylene glycol (PEG) and dextran, including the cyclodextrans,allow for an improve pharmacokinetic profile, resulting in a decrease ininventive peptide clearance.

Intravitreal or intra-aqueous administration of an inventive peptideagent may be indicated in treatment of ocular inflammation, ocularinfection (bacterial, fungal or viral) and the glaucomatous eye bycontrolling F-actin architecture in outflow pathway cells. (Read A T etal., Exp Eye Res, 2006 June:82(6):974-85).

A Preferred Injectable Formulation: Each ml of the peptide agentcomprising amino acid sequence LKKTET [SEQ ID NO:1] or LKKTNT [SEQ IDNO:2] or a conservative variant contains:

Peptide at a concentration of from about 0.001 to 1,000 mg/ml,preferably from about 0.01 mg/ml to 600 mg/ml, more preferably peptideat a concentration of about 0.1 mg/ml to 60 mg/ml, most preferablypeptide at a concentration of about 1 mg/ml to 6 mg/ml.

Preferred Carrier/Vehicle (1): 20 mM Sodium citrate; 50 mM glycine; 3%sucrose; NaOH or HCl to adjust pH; Water for Injection, USP.

Preferred Carrier/Vehicle (2): Peptide encapsulation in PLGAmicrospheres; 20 mM Sodium citrate; 50 mM glycine; 3% sucrose; NaOH orHCl to adjust pH; Water for Injection, USP.

Exemplary Formulation Dosing:

Topical Solutions and Suspensions:

One drop per administration and at least five minutes betweenadministrations is recommended. Immediately after instilling a drop onthe eye, place pressure on the lacrimal sac for one or two minutes toreduce the rate of drug loss through this pathway. Injectable Dosing Use27-30 gauge needle, 0.5 inch length.

A composition according to one embodiment may be in lyophilized form, orin a form capable of being lyophilized, comprising a peptide agentcomprising amino acid sequence LKKTET [SEQ ID NO:1] or LKKTNT [SEQ IDNO:2], a conservative variant thereof, or a stimulating agent thatstimulates production of an LKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO:2]peptide, or a conservative variant thereof, the composition furthercomprising at least one amino acid stabilizing agent. The compositionmay comprise a peptide agent comprising amino acid sequence LKKTET [SEQID NO:1] or LKKTNT [SEQ ID NO:2], a conservative variant thereof, or astimulating agent that stimulates production of an LKKTET [SEQ ID NO:1]or LKKTNT [SEQ ID NO:2] peptide, or a conservative variant thereof, andat least one of a lyophilization bulking agent or an amino acidstabilizing agent, said composition being in lyophilized form. Thecomposition may further comprise at least one of an acidic or basic pHadjusting agent capable of adjusting pH of the composition to a desiredphysiologically acceptable pH level in an aqueous medium, and a bufferto substantially maintain said desired pH in said aqueous medium. Theamino acid stabilizing agent may comprise at least one of alanine,lysine, glycine or glutamic acid. The amino acid stabilizing agent maycomprise at least one 50 mM amino acid stabilizer. The amino acidstabilizing agent may comprise 50 mM glycine. The composition mayfurther comprise a bulking agent comprising at least one ofcarbohydrates, sugar alcohols, mono-, di-, and poly-saccharides,polyols, mannitol, sorbitol, glycerol, sucrose or dextrose. The pHadjusting agent may include at least one of NaOH or HCl. The buffer mayinclude at least one of a sodium salt of at least one of acetic,ascorbic, boric, carbonic, phosphoric, citric, gluconic, lactic orpropionic acids, a calcium salt of carbonic or propionic acids, apotassium salt of phosphoric acid, Tris buffer, acetate, phosphate,citrate or borate buffers. The buffer may be sodium citrate. The buffermay have a buffer capacity of about 0.05-1.0. The buffer may have abuffer capacity of about 0.02-0.2, or about 0.01-0.1. The desired pHlevel may be within a range of about 3.5-11.5, about 3.5-9, about 4.5-8,or about 5.0-7.8. The desired pH level may be about 5.5. The peptideagent may comprise amino acid sequence KLKKTET [SEQ ID NO:3], amino acidsequence LKKTETQ [SEQ ID NO:4], Tβ34, an N-terminal variant of Tβ4, aC-terminal variant of Tβ4, or an isoform of Tβ4. The composition mayfurther include an aqueous medium, wherein said peptide agent is presentin said aqueous medium at a concentration within a range of about0.001-1,000 mg/ml. The composition may further include at least onesteroid.

A composition according to another embodiment is for administration toskin tissue of a subject, and comprises a peptide agent comprising aminoacid sequence LKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO:2], aconservative variant thereof, or a stimulating agent that stimulatesproduction of an LKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO:2] peptide,or a conservative variant thereof, in said tissue, the compositionfurther comprising a quaternary ammonium salt and a topical carrier forapplication to skin tissue of said subject. The peptide agent maycomprise amino acid sequence LKKTET [SEQ ID NO:1], amino acid sequenceLKKTETQ [SEQ ID NO:4], Tβ4, an N-terminal variant of Tβ4, or an isoformof Tβ4. The quaternary ammonium salt may comprise benzalkonium chloride.The peptide agent may be at a concentration of about 0.001-1,000 mg/mland said quaternary ammonium salt may be present in said composition atabout 0.001-1% by weight. The composition may be in a form of asolution, gel, cream, paste, lotion, spray, suspension, dispersion,salve, hydrogel, ointment or foam formulation. The composition may be acosmetic formulation.

A pharmaceutical or cosmetic combination according to another embodimentcomprises a peptide agent comprising amino acid sequence LKKTET [SEQ IDNO:1] or LKKTNT [SEQ ID NO:2], a conservative variant thereof, or astimulating agent that stimulates production of an LKKTET [SEQ ID NO:1]or LKKTNT [SEQ ID NO:2] peptide, or a conservative variant thereof, thecombination further comprising a quaternary ammonium salt, wherein saidagent and said salt can be administered to a subject separately ortogether. The peptide agent may comprise amino acid sequence LKKTET [SEQID NO:1], amino acid sequence LKKTETQ [SEQ ID NO:4], Tβ4, an N-terminalvariant Tβ4, or an isoform of Tβ4. The quaternary ammonium salt may bebenzalkonium chloride. The combination may comprise a pharmaceutical,ophthalmic or cosmetic composition comprising said peptide agent at aconcentration of about 0.001-1,000 mg/ml, and wherein said quaternaryammonium salt may be present in said composition at about 0.0001-1% byweight. The combination may comprise an ophthalmic composition furthercomprising an opthalmically acceptable carrier. The composition maycomprise an eye drop composition.

According to a further embodiment, method of treatment for treating,preventing, inhibiting or reducing tissue deterioration, injury ordamage resulting from administration of a quaternary ammonium salt to asubject, comprises administering said quaternary ammonium salt to saidsubject, and administering to said subject a peptide agent comprisingamino acid sequence LKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO:2], aconservative variant thereof, or a stimulating agent that stimulatesproduction of an LKKTET [SEQ ID NO:1] or LKKTNT [SEQ ID NO:2] peptide,or a conservative variant thereof in said tissue. The agent may beadministered to said subject prior to, concurrently with or after saidadministration of said quaternary ammonium salt. The agent and said saltmay be administered together as a composition. The composition mayfurther comprise an opthalmically acceptable carrier. The compositionmay comprise an eye drop composition. The composition may comprise acosmetically acceptable carrier. The composition may be in a form of asolution, gel, cream, past, lotion, spray, suspension, dispersion,salve, hydrogel, ointment or foam formulation. The peptide agent maycomprise amino acid sequence KLKKTET [SEQ ID NO:3], amino acid sequenceLKKTETQ [SEQ ID NO:4], Tβ4, an N-terminal variant of Tβ4, a C-terminalvariant of Tβ4, or an isoform of Tβ4. The quaternary ammonium salt maycomprise benzalkonium chloride. The peptid agent may be included in saidcomposition at a concentration within a range of about 0.001-1,000mg/ml, and said quaternary ammonium salt may be present in saidcomposition within a range of about 0.0001-1% by weight.

A composition according to still a further embodiment comprises apeptide agent comprising amino acid sequence LKKTET [SEQ ID NO:1] orLKKTNT [SEQ ID NO:2], a conservative variant thereof, or a stimulatingagent that stimulates production of an LKKTET [SEQ ID NO:1] or LKKTNT[SEQ ID NO:2] peptide, or a conservative variant thereof, and anopthalmically acceptable carrier, an antimicrobially effectivepreservative, a tonicity agent for providing said composition with anopthalmically acceptable tonicity, a comfort enhancing agent, at leastone of an acidic or basic pH adjusting agent capable of adjusting pH ofthe composition to a desired ophthalmically acceptable pH level, and abuffer to substantially maintain said desired pH level. The compositionmay further comprise at least one of an antioxidant or an oxygensequestering agent. The antioxidant or oxygen sequestering agent maycomprise at least one of sodium sulfite, sodium thiosulfite, sodiumbisulfite, sodium metabisulfite, thiourea, ascorbic acid, EDTA/disodiumedentate, acetic acid, citric acid, glutathione, acetylcysteine ormethionine. The antioxidant or oxygen sequestering agent may be at aconcentration in said composition within a range of about 0.0001-1.0% byweight. The antimicrobially effective preservative may comprise at leastone of a quaternary ammonium compound, benzalkonium chloride,benzethonium chloride, cetalkonium chloride, cetrimide, benzododeciniumbromide, benzoxonium chloride, an alkyl-mercury salt of thiosalicylicacid, thimerosal, phenylmercuric nitrate, phenylmercuric acetate,phenylmercuric borate, a paraben, methylparaben, propylparaben, achelating agent, disodium edentate, sodium gluconate, sodium propionate,an alcohol, chlorobutanol, benzyl alcohol, phenyl ethanol, a guanidinederivative, chlorohexidine, polyhexamethylene biguanide, sorbic acid,boric acid, chlorine dioxide, polyquatemium or myristamidopropyldiethylamine. The preservative may be present in said composition at aconcentration within a range of about 0.0001-5.0% (w/v). The compositionmay further include at least one opthalmically acceptable stabilizingagent. The stabilizing agent may comprise at least one of a polyol,mannitol, sorbitol, glycerol, sucrose, an amino acid stabilizer, alanine(Ala), lysine (Lys), glycine (Gly) or glutamic acid (Glu). Thestabilizing agent may be present in said composition at a concentrationwithin a range of about 0.01-10% by weight. The tonicity agent maycomprise at least one of an ionic compound, alkali metal halide,alkaline earth metal halide, CaCl2, KBr, KCl, LiCl, NaBr, NaCl, boricacid, a non-ionic compound, urea, glycerol, sorbitol, mannitol,propylene glycol, or dextrose. The composition may have an osmolalitywithin a range of about 50 to 1000 mOsmol. The adjusting agent mayinclude at least one of NaOH or HCL. The buffer may include at least oneof a sodium salt of acetic acid, a sodium salt of ascorbic acid, asodium salt of boric acid, a sodium salt of carbonic acid, a sodium saltof phosphoric acid, a sodium salt of citric acid, a sodium salt ofgluconic acid, a sodium salt of lactic acid, a sodium salt of propionicacid, a calcium salt of carbonic acid, a calcium salt of propionic acid,a potassium salt of phosphoric acid, Tris buffer, an acetate buffer, aphosphate buffer, a citrate buffer or a borate buffer. The desired pHlevel may be within a range of about 3.5-11.5. The buffer may have abuffer capacity of about 0.05-1.0. The peptide agent may comprise aminoacid sequence KLKKTET [SEQ ID NO:3], amino acid sequence LKKTETQ [SEQ IDNO:4], Tβ4, an N-terminal variant of Tβ4, a C-terminal variant of Tβ4,or an isoform of Tβ4. The composition may further comprise an aqueousmedium, wherein said peptide agent is present in said aqueous medium ata concentration within a range of about 0.001-1,000 mg/ml. Thecomposition may be in a form of a solution, gel, cream, paste, lotion,spray, suspension, dispersion, salve, hydrogel, ointment or foamformulation. The composition may comprise an eye drop composition. Thepeptide agent may be present in said composition encapsulated with atleast one of phospholipid membranes, liposomes, microspheres,nanoparticles or biodegradable polymers, or as a peptide-polymerconjugate.

Example

Thymosin beta 4 (Tβ₄), a 43 amino acid molecule, promotes ocular woundhealing, decreases ocular inflammation, and has anti-apoptotic effectson corneal epithelium. In this study, the effect of Tβ₄ on the survivalof cultured human corneal epithelial cells exposed to benzalkoniumchloride (BAK) was measured.

Human corneal epithelial cells at approximately 80% confluence weretreated with 0%, 0.001%, 0.01%, or 0.1% BAK for 15 minutes. After 3 and24 hours of recovery in culture medium, cell proliferation was measuredusing a colorimetric BrdU incorporation assay. Apoptosis was measuredusing a colorimetric annexin-based cell death assay. Studies wererepeated in the presence of 1 mcg/ml Tβ₄, an in vitro dosagedemonstrated effective in several published studies. To further assessthe ability of Tb₄ to prevent apoptosis, corneal epithelial cells weretreated with 0.01% BAK±Tβ₄ over a 5 day time course.

At all BAK concentrations used, corneal epithelial cell proliferationwas inhibited, and apoptosis was increased, compared to control at 3 and24 hours recovery time. At the 3 and 24 hour time points, Tβ₄ did notabrogate the deleterious effects of BAK; cell proliferation was notpromoted by Tb₄ and apoptosis was not inhibited. However, at longertimes in culture (2 to 5 days), Tβ₄ treatment significantly inhibitedthe BAK-initiated epithelial cell apoptosis. In addition, Tβ₄-treatedcells demonstrated decreased apoptosis compared to those cultured inmedium alone for 5 days.

BAK, a preservative used in many commercially available ocularsolutions, induces corneal epithelial cell apoptosis in culture,suggesting that long-term exposure is deleterious to corneal health. Thestudy reported here indicates that Tβ₄ is able to overcome thedeleterious pro-apoptotic effects of BAK. Since many BAK-containing eyedrops are typically used for extended periods of time, Tβ₄ is indicatedto be a useful additive to solutions containing this preservative.

1. A method of treating elevated intraocular pressure in a subjecthaving elevated intraocular pressure, comprising administering to thesubject an ophthalmically acceptable composition comprising a peptideagent comprising amino acid sequence LKKTET (SEQ ID NO:1) or Thymosin β4(Tβ4).
 2. The method of claim 1 wherein said composition comprises anaqueous medium.
 3. The method of claim 2, wherein said peptide agent ispresent in said aqueous medium at a concentration within a range ofabout 0.001-1,000 mg/ml.
 4. The method of claim 1 wherein said peptideagent comprises Tβ4.
 5. The method of claim 1 for treatment of glaucomain said subject.
 6. The method of claim 1 wherein said elevatedintraocular pressure is due to use of corticosteroids.